Ig-transgenic MD4 (Jax 2595) mice were originally
purchased from the Jackson Laboratory. Cd79acre/cre
mice on the B6 background were kindly provided
as a gift by M. Reth. The Efnb1fl/fl line was backcrossed to B6 for more than 10 generations before
inter-breeding with other relevant alleles on the
B6 background. Relevant mice were interbred to
obtain dsRed-expressing OT-II mice, CFP-expressing
MD4 mice, Cd79a+/creEfnb1fl/y or Cd79a+/creEfnb1fl/fl
B6 mice, CFP-expressing Cd19+/creEfnb1fl/y or
Cd19+/creEfnb1fl/fl MD4 mice. All mice were maintained under specific pathogen-free conditions,
and used in accordance of governmental and institutional guidelines for animal welfare.

Immunization

For sheep red blood cell (SRBC, Zhengzhou
Baiji, China) immunization, 1 ml of SRBC suspension was washed three times in cold phosphate-buffered saline (PBS) and intraperitioneally
injected into each mouse in a volume of 300 ml.
For some experiments, mice were immunized
intraperitoneally with 100 mg of NP-KLH (NP20
or higher conjugate ratios, Biosearch Technologies) mixed with 1 mg of lipopolysaccharide
(LPS) (Sigma) in alum (Thermo Scientific). For
measuring GC responses by MD4 and OT-II cells,

30 mg of HEL-OVA conjugate antigen made bythe Solulink cross-linking kit (17) or 130 mg ofconjugate antigen made by glutaraldehyde (57)was mixed with 1 mg of LPS in alum and thenused to subcutaneously immunize the mice.

Cell isolation, culture and
retroviral transduction

B and CD4 T lymphocytes were isolated from
pooled spleens and lymph nodes by CD19 or CD4
Microbeads (Miltenyi Biotec) and activated with
1 mg/ml LPS or plate-bound anti-CD3 and anti-
CD28 (BioXcell), respectively. One or 2 days after
activation, T or B cells were spin-infected (1500g)
in the six-well plate at 7.5 × 106 cells per well with
appropriate viral supernatants in the presence of
1 mg/ml polybrene (Sigma) for 2hours at 32°C.
Infected T cells were expanded in 40 U/ml IL-2
(Peprotech) for 3 days before being used for adoptive transfer. Infected B cells were directly used
for conjugated assay.

To measure T cell dynamics by live imaging or distribution in and around EFNB1-deficient or -sufficient
GCs by static imaging, 8 × 105 Cd19+/creEfnb1fl/y or
Cd19+/creEfnb1+/y nonfluorescent or CFP-expressing
MD4 B cells were cotransferred with 105
GFP-expressing OT-II cells to B6 recipients, which were
then immunized as described above. When retrovirally transduced OT-II T cells were involved, GFP-or Ametrine-expressing T cells were enriched by
flow sorting of Ficoll-isolated live cells of the infected T cell culture. Sorted cells were rested in
complete RPMI-1640 for 2 hours at 37°C before
intravenous injection of 106 transduced OT-II
cells and 5 × 105 MD4 B cells per B6 recipient.
These cells were rested in recipient mice for at
least 1 day before HEL-OVA immunization as
described above.